Methods for Purification of Messenger RNA

ABSTRACT

The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of subjecting an impure preparation comprising in vitro synthesized mRNA to a denaturing condition, and purifying the mRNA from the impure preparation from step (a) by tangential flow filtration, wherein the mRNA purified from step (b) is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/784,996, filed Mar. 14, 2013, the entirety ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Messenger RNA therapy is becoming an increasingly important approach forthe treatment of a variety of diseases. Messenger RNA therapy involvesadministration of messenger RNA (mRNA) into a patient in need of thetherapy and production of the protein encoded by the mRNA within thepatient body. Thus, it is important to ensure the production of highlypure and safe mRNA product. Traditionally, RNA purification typicallyemploys spin columns and involves the use of caustic or flammablesolvents, such as ethanol, which is undesirable for therapeuticadministration and large scale production.

SUMMARY OF THE INVENTION

The present invention provides improved methods of purifying mRNA thatis suitable for administration as a pharmaceutical product based ontangential flow filtration (TFF). Prior to the present invention, RNApurification typically employs spin columns and involves the use ofcaustic or flammable solvents, such as ethanol, which is undesirable fortherapeutic administration and large scale production. Further, theprior art method typically does not allow for the separation ofincomplete transcripts known as premature aborts or “shortmers,” whichis reported to be highly immunostimulatory and the presence of which maygreatly alter the toxicity and tolerability profile of mRNA as activepharmaceutical ingredient (API). The present invention is, in part,based on the discovery that tangential flow filtration is surprisinglyeffective to remove reactants, enzymes, by products, in particular, theshortmers, from mRNA production mixture. As described herein, tangentialflow filtration, particularly in combination with a pre-treatment usinga denaturing agent, can effectively remove reactants, enzymes andbyproducts including prematurely aborted RNA sequences (i.e.,shortmers), while still maintaining the integrity of mRNA. Moresurprisingly, the present inventors have demonstrated that tangentialflow filtration can be successfully performed using only aqueous buffersas solvents without using any caustic or flammable solvents. Thus, thepresent invention provides a more effective, reliable, and safer methodof purifying mRNA from large scale manufacturing process therapeuticapplications.

In one aspect, the present invention provides, among other things,methods of purifying messenger RNA (mRNA) including the steps of (a)subjecting an impure preparation comprising in vitro synthesized mRNA toa denaturing condition, and (b) purifying the mRNA from the impurepreparation from step (a) by tangential flow filtration, wherein themRNA purified front step (b) is substantially free of prematurelyaborted RNA sequences and/or enzyme reagents used in in vitro synthesis.

In some embodiments, step (a) comprises adding a protein denaturingagent to the impure preparation. In some embodiments, step (a) comprisesincubating the impure preparation with the protein denaturing agentadded at room temperature for about 1-10 minutes (e.g., about 2-9, 2-8,2-7, 3-10, 3-9, 3-8, 3-7, 3-6, 4-10, 4-9, 4-8, 4-7, 4-6 minutes). Insome embodiments, step (a) comprises incubating the impure preparationwith the protein denaturing agent added at room temperature for about 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, step (a)comprises incubating the impure preparation with the protein denaturingagent added at room temperature for about 5 minutes. In someembodiments, a suitable protein denaturing agent is selected from thegroup consisting of urea, guanidinium thiocyanate, KCl, sodium dodecylsulfate, sarcosyl, other detergents, and combinations thereof.

In sonic embodiments, step (a) comprises adding urea to the impurepreparation to achieve a resulting urea concentration of about 1 M orgreater. In some embodiments, the resulting urea concentration is about2 M or greater, 3 M or greater, 4 M or greater, 5 M or greater, 6 M orgreater, 7 M or greater, 8 M or greater, 9 M or greater, or 10 M orgreater.

In some embodiments, step (a) comprises adding guanidinium thiocyanateto the impure preparation to achieve a resulting guanidinium thiocyanateconcentration of about 1 M or greater. In some embodiments, theresulting guanidinium thiocyanate concentration is about 2 M or greater,3 M or greater, 4 M or greater, 5 M or greater, 6 M or greater, 7 M orgreater, 8 M or greater, 9 M or greater, or 10 M or greater.

In some embodiments, step (a) comprises adding KCl to the impurepreparation to achieve a resulting KCl concentration of about 1 M orgreater. In some embodiments, the resulting KCl concentration is about 2M or greater, 3 M or greater, 4 M or greater, or 5 M or greater.

In some embodiments, the tangential flow filtration is performed usingonly aqueous solvents. In some embodiments, the tangential flowfiltration is performed using water as solvent. In some embodiments, thetangential flow filtration is performed at a feed rate of approximately100-200 mL/minute (e.g., approximately 100-180 mL/minute, 100-160mL/minute, 100-140 mL/minute, 110-190 mL/minute, 110-170 mL/minute, or110-150 mL/minute) and/or a flow rate of approximately 10-50 mL/minute(e.g., approximately 10-40 mL/minute, 10-30 mL/minute, 20-50 mL/minute,or 20-40 mL/minute). In some embodiments, the tangential flow filtrationis performed at a feed rate of approximately 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 mL/minute and/or a flow rate ofapproximately 10, 20, 30, 40, or 50 mL/minute.

In some embodiments, the mRNA purified from step (b) contains less thanabout 5% (e.g., less than about 4%, 3%, 2%, or 1%) of prematurelyaborted RNA sequences and/or enzyme reagents used in in vitro synthesis.In some embodiments, the mRNA purified from step (b) contains less thanabout 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, or 0.1%) of prematurely aborted RNA sequences and/or enzymereagents used in in vitro synthesis. In some embodiments, the mRNApurified from step (b) contains less than 0.5% of prematurely abortedRNA sequences and/or enzyme reagents used in in vitro synthesis. In someembodiments, the mRNA purified from step (b) contains less than 0.1% ofprematurely, aborted RNA sequences and/or enzyme reagents used in invitro synthesis. In some embodiments, the mRNA purified from step (b)contains undetectable prematurely aborted RNA sequences and/or enzymereagents used in in vitro synthesis as determined by eithidium bromideand/or Coomassie staining.

In some embodiments, the prematurely aborted RNA sequences comprise lessthan 15 bases (e.g., less than 14, 13, 12, 11, 10, 9 or 8 bases). Insome embodiments, the prematurely aborted RNA sequences comprise about8-12 bases.

In some embodiments, the enzyme reagents used in in synthesis compriseT7 RNA polymerase, DNAse I, pyrophosphatase, and/or RNAse inhibitor. Insome embodiments, the enzyme reagents used in in vitro synthesiscomprise T7 RNA polymerase.

In some embodiments, the tangential flow filtration is performed beforea cap and poly-A tail are added to the in vitro synthesized mRNA. Insome embodiments, the tangential flow filtration is performed after acap and poly-A tail are added to the in vitro synthesized mRNA. In someembodiments, the tangential flow filtration is performed both before andafter a cap and poly-A tail are added to the in vitro synthesized mRNA.

In some embodiments, the in vitro synthesized mRNA is greater than about1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb inlength. In some embodiments, the in vitro synthesized mRNA comprises oneor more modifications to enhance stability. In some embodiments, the oneor more modifications are selected from modified nucleotide, modifiedsugar phosphate backbones, 5′ and/or 3′ untranslated region. In someembodiments, the in vitro synthesized mRNA is unmodified.

In some embodiments, the mRNA purified from step (b) has an integritygreater than about 95% (e.g., greater than about 96%, 97%, 98%, 99% ormore). In some embodiments, the mRNA purified from step (b) has anintegrity greater than 98%. In some embodiments, the mRNA purified fromstep (b) has an integrity greater than 99%. In some embodiments, themRNA purified from step (b) has an integrity of approximately 100%.

The present invention also provides methods for manufacturing messengerRNA (mRNA) including the steps of synthesizing mRNA in vitro, andpurifying the in vitro synthesized mRNA according to methods describedherein.

The present invention also provides messenger RNA (mRNA) purifiedaccording to the methods described herein.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The following figures are for illustration purposes only and not forlimitation.

FIG. 1 shows exemplary protein levels in in vitro transcription of FFLmRNA samples purified according to provided methods, including exposureto urea, along with various controls as shown by gel electrophoresis andCoomassie staining.

FIG. 2 shows exemplary firefly luciferase (FFL) mRNA levels in in vitrotranscription samples purified according to provided methods as comparedto mRNA purified according to traditional methods as shown by agarosegel electrophoresis and ethidium bromide staining.

FIG. 3 shows exemplary protein levels in in vitro transcription samplesof FFL mRNA purified according to provided methods, including TFF withand without exposure to 5M urea, as compared to mRNA purified accordingto traditional methods gel electrophoresis and Coomassie staining.

FIG. 4 depicts exemplary fluorescence data gathered from translatedpurified FFL mRNA provided from provided methods as compared to purifiedmRNA provided from traditional methods.

FIG. 5 shows exemplary protein levels from in vitro transcriptionsamples of Factor IX (FIX) mRNA purified, according to provided,methods, including exposure to proteinase K and/or 5M Urea, as comparedto mRNA purified according to traditional methods gel electrophoresisand Coomassie staining.

FIG. 6 shows exemplary FIX mRNA levels in in vitro transcription samplespurified according to provided methods as shown by agarose gelelectrophoresis and ethidium bromide staining.

FIG. 7 shows exemplary protein levels in in vitro transcription samplesof cystic fibrosis transmembrane conductance regulator (CCPR) mRNApurified according to provided methods, including exposure to 2M KCl, ascompared to mRNA purified according to traditional methods gelelectrophoresis and Coomassie staining.

FIG. 8 shows exemplary CFTR mRNA levels in in vitro transcriptionsamples purified according to provided methods, including exposure to 2MKCl, as shown by agarose gel electrophoresis and ethidium bromidestaining.

FIG. 9 shows exemplary CFTR mRNA levels in in vitro transcriptionsamples purified according to provided methods, including exposure to 2MKCl, as compared to mRNA purified according to traditional methods asshown by agarose gel electrophoresis and ethidium bromide staining.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide (e.g., heavy chainor light chain of antibody), assemble multiple polypeptides (e.g., heavychain or light chain of antibody) into an intact protein (e.g.,antibody) and/or post-translational modification of a polypeptide orfully assembled protein (e.g., antibody). In this application, the terms“expression” and “production,” and grammatical equivalent, are usedinter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule m a form in which it exhibits a property and/oractivity by which it is characterized,

improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

Impurities: As used herein, the term “impurities” refers to substancesinside a confined amount of liquid, gas, or solid, which differ from thechemical composition of the target material or compound impurities arealso referred to as contaminants.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

isolated: As used herein, the term “isolated” refers to a substanceand/or entity, that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

messenger RNA (mRMA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide, mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions.

mRNA integrity: As used herein, the term “mRNA integrity” generallyrefers to the quality of mRNA. In some embodiments, mRNA integrityrefers to the percentage of mRNA that is not degraded after apurification process (e.g., tangential flow filtration). mRNA integritymay be determined using methods well known in the art, for example, byRNA agarose gel electrophoresis (e.g., Ausubel et al., John Weley &Sons, Inc., 1997, Current Protocols in Molecular Biology).

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g, nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In sonic embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA. Furthermore, theterms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleicacid analogs, i.e., analogs having other than a phosphodiester backbone.For example, the so-called “peptide nucleic acids,” which are known inthe art and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. Theterm “nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleotide sequences that encodeproteins and/or RNA may include introns. Nucleic acids can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine);chemically modified bases; biologically modified bases (e.g., methylatedbases); intercalated bases; modified sugars (e.g., 2′-fluororibose,ribose 2′-deoxyribose, arabinose, and hexose); and/or modified phosphategroups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). Insome embodiments, the present invention is specifically directed to“unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotidesand residues, including nucleotides and/or nucleosides) that have notbeen chemically modified in order to facilitate or achieve delivery.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., firexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Prematurely aborted RATA sequences: The term “prematurely aborted RNAsequences”, as used herein, refers to incomplete products of an mRNAsynthesis reaction (e.g., an in vitro synthesis reaction). For a varietyof reasons, RNA polymerases do not always complete transcription of aDNA template; i.e., RNA synthesis terminates prematurely. Possiblecauses of premature termination of RNA synthesis include quality of theDNA template, polymerase terminator sequences for a particularpolymerase present in the template, degraded buffers, temperature,depletion of ribonucleotides, and mRNA secondary structures. Prematurelyaborted RNA sequences may be any length that is less than the intendedlength of the desired transcriptional product. For example, prematurelyaborted mRNA sequences may be less than 1000 bases, less than 500 bases,less than 100 bases, less than 50 bases, less than 40 bases, less than30 bases, less than 20 bases, less than 15 bases, less than 10 bases orfewer.

Salt: As used herein the terns “salt” refers to an ionic compound thatdoes or may result from a neutralization reaction between an acid and abase.

Subject: As used herein, the term “subject” refers to a human or anynon-human (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep,horse or primate). A human includes pre- and post-natal forms. In manyembodiments, a subject is a human being. A subject can be a patient,which refers to a human presenting to a medical provider for diagnosisor treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially free: As used herein, the term “substantially free” refersto a state in which relatively little or no amount of a substance to beremoved (e.g., prematurely aborted RNA sequences) are present. Forexample, “substantially free of prematurely aborted RNA sequences” meansthe prematurely aborted RNA sequences are present at a level less thanapproximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1% or less (w/w) of the impurity. Alternatively,“substantially free of prematurely aborted RNA sequences” means theprematurely aborted RNA sequences are present at a level less than about100 ng, 90 ng, 80 ng, 70 ng, 60 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ηg, 1ηg, 500 ρg, 100 ρg, 50 ρg, 10 ρg, or less.

DETAILED DESCRIPTION

The present invention provides, among other things, improved methods forpurifying mRNA from an impure preparation (e.g., in vitro synthesisreaction mixture) based on tangential flow filtration. In someembodiments, an inventive method according to the present inventionincludes steps of (a) subjecting an impure preparation comprising invitro synthesized mRNA to a denaturing condition, and (b) purifying themRNA from the impure preparation from step (a) by tangential flowfiltration, wherein the mRNA purified from step (b) is substantiallyfree of prematurely aborted. RNA sequences and/or enzyme reagents usedin in vitro synthesis.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Synthesis of mRNA

mRNA is typically thought of as the type of RNA that carries informationfrom DNA to the ribosome. The existence of mRNA is typically very briefand includes processing and translation, followed by degradation.Typically, in eukaryotic organisms, mRNA processing comprises theaddition of a “cap” on the N-terminal (5′) end, and a “tail” on theC-terminal (3′) end. A typical cap is a 7-methylguanosine cap, which isa guanosine that is linked through a 5′-5′-triphosphate bond to thefirst transcribed nucleotide. The presence of the cap is important inproviding resistance to nucleases found in most eukaryotic cells. Thetail is typically a polyadenylation event whereby a polyadenylyl moietyis added to the 3′ end of the mRNA molecule. The presence of this “tail”serves to protect the mRNA from exonuclease degradation. Messenger RNAis translated by the ribosomes into a series of amino acids that make upa protein.

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7, SP6 RNA polymerase), DNAse I,pyrophosphatase, andlor RNAse inhibitor. The exact conditions will varyaccording to the specific application. The presence of these reagents isundesirable in the final product according to several embodiments andmay thus be referred to as impurities and a preparation containing oneor more of these impurities may be referred to as an impure preparation.

mRNAs according to the present invention may be purified on a commercialscale. In some embodiments, the mRNA is purified at a scale of orgreater than 0.1 grams, 0.5 grams, 1 gram, 2 grams, 3 grams, 4 grams, 5grams, 6 grams, 7 grams, 8 grams, 9 grams, 10 gram, 20 grams, 30 grams,40 grams, 50 grams, 60 grams, 70 grams, 80 grams, 90 grams, 100 grams,200 grams, 300 grams, 400 grams, 500 grams, 600 grams, 700 grams, 800grams, 900 grams, or 1,000 grams per batch.

According to various embodiments, the present invention may be used topurify in vitro synthesized mRNA of a variety of lengths. In someembodiments, the present invention may be used to purify in vitrosynthesized mRNA of greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb,3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb,13 kb, 14 kb, or 15 kb in length. In some embodiments, the presentinvention may be used to purify mRNA containing one or moremodifications that typically enhance stability. In some embodiments, oneor more modifications are selected from modified nucleotide, modifiedsugar phosphate backbones, 5′ and/or 3′ untranslated region. In someembodiments, the present invention may, be used to purify in vitrosynthesized mRNA that is unmodified.

Typically, mRNAs are modified to enhance stability. Modifications ofmRNA can include, for example, modifications of the nucleotides of theRNA. An modified mRNA according to the invention can thus include, forexample, backbone modifications, sugar modifications or basemodifications. In some embodiments, antibody encoding mRNAs (e.g., heavychain and light chain encoding mRNAs) may be synthesized from naturallyoccurring nucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purities (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066,4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319,5,262,530 and 5,700,642, the disclosure of which is included here in itsfull scope by reference.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs include a 5′ cap structure. A 5′ cap istypicallyadded as follows: first, an RNA terminal phosphatase removesone of the terminal phosphate groups from the 5′ nucleotide, leaving twoterminal phosphates; guanosine triphosphate (GTP) is then added to theterminal phosphates via a guanylyl transferase, producing a 5′5′5triphosphate linkage; and the 7-nitrogen of guanine is then methylatedby a methyltransferase. Examples of cap structures include, but are notlimited to, m7G(5)ppp (5′(A, G(5′)ppp(5′)A and G(5′)ppp(5′)G.

While mRNA provided from in vitro transcription reactions may bedesirable in some embodiments, other sources of mRNA are contemplated aswithin the scope of the invention including wild-type mRNA producedfront bacteria, fungi, plants, and/or animals.

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRINA's stability or translation, for example,an iron responsive element. In some embodiments, a 5′ untranslatedregion may be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anMRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

The present invention may be used to purify mRNAs encoding a variety ofproteins. Non-limiting examples of purification of mRNAs encodingfirefly luciferase, Factor IX, and CTIR, are described in detail in theExamples section.

Denaturing Conditions and Denaturation Agents

Typically, changing the conformation of a protein or nucleic acid eithertemporarily or permanently by disrupting intermolecular forces is calleddenaturation. Denaturation results in structural change and often to aloss of activity. Since the native conformation of a molecule is usuallythe most water soluble, disrupting the secondary and tertiary structuresof a molecule may cause changes in solubility and may result inprecipitation of the protein or nucleic acid from solution.Surprisingly, as described herein, using a denaturing condition incombination with tangential flow filtration (TFF) can facilitate mRNApurification while still maintaining the integrity of mRNA.

As used herein, the term “denaturing condition” refers to any chemicalor physical conditions that can cause denaturation. Exemplary denaturingconditions include, but are not limited to, chemical reagents, hightemperatures, extreme pH, etc.

In some embodiments, a denaturing condition is achieved through addingone or more denaturing agents to an impure preparation containing mRNAto be purified. In some embodiments, a denaturing agent suitable for thepresent invention is a protein and/or DNA denaturing agent. In someembodiments, a denaturing agent may be: 1) an enzyme (such as a serineproteinase or a DNase), 2) an acid, 3) a solvent, 4) a cross-linkingagent, 5) a chaotropic agent, 6) a reducing agent, and/or 7) high ionicstrength via high salt concentrations. In some embodiments, a particularagent may fall into more than one of these categories.

In some embodiments, one or more enzymes may be used as denaturingagents to degrade proteins and DNA templates used in mRNA synthesis. Insome embodiments, suitable enzymes include, but are not limited to,serine proteases such as chymotrypsin and chymotrypsin-like serineproteases, trypsin and trypsin-like serine proteases, elastase andelastase-like serine proteases, subtilisin and subtilisin-like serineproteases, and combinations thereof, deoxyribonucleases (DNases) such asdeoxyribonuclease I, II and/or IV, restriction enzymes such as EcoRI,EcoRII, BamHI, HindIII, SpeI, SphI, StuI, XbaI, and combination thereof.

In some embodiments, an acid may be used as a denaturing agent. In someembodiments, a suitable acid may be acetic acid, formic acid, oxalicacid, citric acid, benzoic acid, chloroacetic acid, dichloroacetic acid,trichloroacetic acid, ascorbic acid, sulfosalicylic acid, andcombinations thereof.

In some embodiments, a solvent may be used as a denaturing agent. Insome embodiments, a solvent may be isopropyl alcohol, acetone, methylethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatoniurn,and combinations thereof.

In some embodiments, a chaotropic agent may be sued as a denaturingagent. Choatropic agents are substances which disrupt the structure ofmacromolecules such as proteins and nucleic acids by interfering withnon-covalent forces such as hydrogen bonds and van der Waals forces. Insome embodiments, a chaotropic agent may be urea, thiourea, guanidiniumchloride, guanidinium thiocyanate, guanidinium isothiocyanate, lithiumacetate, magnesium chloride, sodium dodecyl sulfate, lithium perchlorateand combination thereof.

In some embodiments, an impure preparation containing mRNA to bepurified is treated with urea. In some embodiments, an amount of urea isadded such that the resulting urea concentration is about 1M or greater.In some embodiments, urea is added such that the resulting ureaconcentration is about 2 M or greater, 3 M or greater, 4 M or greater, 5M or greater, 6 M or greater, 7 M or greater, 8 M or greater, 9 M orgreater, or 10 M or greater. In some embodiments, an impure preparationcontaining mRNA to be purified is treated with guanidinium thiocyanate.In some embodiments, an amount of guanidinium thiocyanate is added suchthat the resulting guanidinium thiocyanate concentration is about 1 M orgreater. In some embodiments, guanidinium thiocyanate is added such thatthe resulting guanidinium thiocyanate concentration is about 2 M orgreater, 3 M or greater, 4 M or greater, 5 M or greater, 6 M or greater,7 M or greater, 8 M or greater, 9 M or greater, or 10 M or greater.

In some embodiments, a reducing agent may be used as a denaturing agent.Reducing agents are compounds that donate an electron to anotherspecies, thus becoming oxidized itself. In some embodiments, a reducingagent may be lithium aluminum hydride, sodium amalgam, diborane, sodiumborohydride, sulfites, diisobutylaluminum hydride, phosphites, carbonmonoxide, 2-mercaptoethanol, dithiothreitol, ortris(2-carboxyethyl)phosphine, and combinations thereof.

In some embodiments, one or more of pH, heat, and/or heavy metals (suchas lead, mercury or cadmium) may also be used a denaturing agents.Extremes of pH are known to cause a protein to denature. Although thebackbone of a protein chain is neutral, the amino acid residues thatcomprise the protein often contain acidic and basic groups. These groupsare usually charged and can form salt bridges with a group of oppositecharge. Accordingly, extremes of pH can change the charges on theseacidic and basic groups, disrupting salt bridges.

In some embodiments, less drastic changes in pH may also affect theactivity and solubility of a protein. Like individual amino acids,proteins have an isoelectric point at which the number of negativecharges equals the number of positive charges. This is frequently thepoint of minimum water solubility. At the isoelectric pH, there is nonet charge on the molecule. Individual molecules have a tendency toapproach one another, coagulate, and precipitate out of solution. At apH above or below the isoelectric pH, the molecules have a net negativeor positive charge, respectively. Thus when proteinmolecules approacheach other, they have the same overall charge and repulse each other.

In some embodiments, heat may be used as a denaturing agent. Heat cansupply kinetic energy to protein molecules, causing their atoms tovibrate more rapidly. In some embodiments, this will disrupt relativelyweak forces such as hydrogen bonds and hydrophobic interactions. Heat isalso used in sterilization to denature and hence destroy the enzymes inbacteria.

In some embodiments, salts of metal ions such as mercury(II), lead(II),and silver may be used as denaturing agents due to their ability to formstrong bonds with disulfide groups and with the carboxylate ions of theacidic amino acids. Thus, they disrupt both disulfide bridges and saltlinkages and cause the protein to precipitate out of solution as aninsoluble metal-protein salt.

In some embodiments, high concentrations of salt (high salinity) mayalso be used as a denaturing agent. High concentrations of salts areknown to cause both proteins and nucleic acids to precipitate from anaqueous solution. In some embodiments, a high concentration of salt maybe between 1M and 10M, inclusive. In some embodiments, a highconcentration of salt may be between 2M and 9M, inclusive. In someembodiments, a high concentration of salt may be between 2M and 8M,inclusive. In some embodiments, a high concentration of salt may bebetween 2M and 5M, inclusive. In some embodiments, a high concentrationof salt may be greater than 1M concentration. In some embodiments, ahigh concentration of salt may be greater than 2M concentration. In someembodiments, a high concentration of salt may be greater than 3Mconcentration. In some embodiments, a high concentration of salt may begreater than 4M concentration. In some embodiments, a high concentrationof salt may be greater than 5M concentration. In some embodiments, ahigh concentration of salt may be greater than 6M concentration. In someembodiments, a high concentration of salt may be greater than 7Mconcentration. In some embodiments, a high concentration of salt may begreater than 8M concentration. In some embodiments, a single salt isused as a denaturing agent. In some embodiments, more than one salt isused as a denaturing agent.

In some embodiments, a salt used as a denaturing agent may be a calciumsalt, an iron salt, a magnesium salt, a potassium salt, a sodium salt,or a combination thereof. Exemplary specific salts suitable for use asdenaturing agents in some embodiments include, but are not limited to,potassium chloride (KCl), sodium chloride (NaCl), lithium chloride(LiCl), calcium chloride (CaCl₂), potassium bromide (KBr), sodiumbromide (NaBr), lithium bromide (LiBr). In some embodiments, thedenaturing agent the impure preparation is subjected to is potassiumchloride (KCl). In some embodiments, KCl is added such that theresulting KCl concentration is about 1M or greater. In some embodiments,KCl is added such that the resulting KCl concentration is about 2 M orgreater, 3 M or greater, 4 M or greater, or 5 M or greater.

In some embodiments, it may be desirable to incubate the impurepreparation with one or more denaturing agents for a period of time. Insome embodiments, the impure preparation is incubated with a denaturingagent for less than one minute. In some embodiments, the impurepreparation is incubated with a denaturing agent for one minute. In someembodiments, the impure preparation is incubated with a denaturing agentfor two minutes. In some embodiments, the impure preparation isincubated with a denaturing agent for three minutes. In someembodiments, the impure preparation is incubated with a denaturing agentfor four minutes. In some embodiments, the impure preparation isincubated with a denaturing agent for five minutes. In some embodiments,the impure preparation is incubated with a denaturing agent for tenminutes. In some embodiments, the impure preparation is incubated with adenaturing agent for one hour. In some embodiments, the impurepreparation is incubated with a denaturing agent for two hours.

In some embodiments, the impure preparation is incubated with one ormore denaturing agents at room temperature (e.g., about 20-25° C.). Insome embodiments, the impure preparation is incubated with one or moredenaturing agents at a temperature below room temperature. In someembodiments, the impure preparation is incubated with one or moredenaturing agents at a temperature above room temperature.

Purification

In several embodiments, before and/or after exposure to a denaturingcondition, tangential flow filtration is used to purify the mRNA from animpure preparation. In some embodiments, tangential flow filtration isperformed before a cap and poly-A tail are added to the in vitrosynthesized mRNA. In some embodiments, tangential flow filtration isperformed after a cap and poly-A tail are added to the in vitrosynthesized mRNA. In some embodiments, tangential flow filtration isperformed both bef=ore and after a cap and poly-A tail are added to thein vitro synthesized mRNA.

Traditional Membrane Filtration

Generally, membrane filtration involves separating solids from fluidsusing one or more interposed permeable membranes. Membrane filtrationmay also be used to filter particles from a gaseous sample. There aretwo major forms of membrane filtration, passive filtration whichproceeds solely due to solution-diffusion, and active filtration whichuses positive pressure or negative pressure (i.e. vacuum) to force theliquid or gas across the membrane.

Traditional membrane filtration is also known as “dead-end” filtration.In this format, the feed is loaded onto a membrane and forced through bypositive or negative pressure. Dead-end filtration tends to beinexpensive and simple, with the major drawbacks being fouling orclogging of the membrane with non- or slowly-permeating solute (alsoreferred to as the retentate), and concentration polarization.Generally, membranes tend to clog or foul more rapidly as driving forcesincrease. As a membrane fouls or clogs, the rate of filtration isreduced and eventually no permeate is able to pass through until thefilter is changed or cleaned. Concentration polarization is a phenomenonwherein non-permeable solute collects on the surface of a filter andeventually forms a type of secondary membrane, which further impedestravel of permeable solute across the membrane. As a result, dead-endfiltration is typically used in batch type processes.

Tangential Flow Filtration

Tangential flow filtration (TFT), also referred to as cross-flowfiltration, is a type of filtration wherein the material to be filteredis passed tangentially across a filter rather than through it. In TFF,undesired permeate passes through the filter, while the desiredretentate passes along the filter and is collected downstream. It isimportant to note that the desired material is typically contained inthe retentate in TFF, which is the opposite of what one normallyencounters in traditional-dead end filtration.

Depending upon the material to be filtered, TFF is usually used foreither microfiltration or ultrafiltration. Microfiltration is typicallydefined as instances where the filter has a pore size of between 0.05 μmand 1.0 μm, inclusive, while ultrafiltration typically involves filterswith a pore size of less than 0.05 μm. Pore size also determines thenominal molecular weight limits (NAM-1), also referred to as themolecular weight cut off (MWCO) for a particular filter, withmicrofiltration membranes typically having NMWLs of greater than 1,000kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1kDa and 1,000 kDa.

A principal advantage of tangential flow filtration is thatnon-permeable particles that may aggregate in and block the filter(sometimes referred to as “filter cake”) during traditional “dead-end”filtration, are instead carried along the surface of the filter. Thisadvantage allows tangential flow filtration to be widely used inindustrial processes requiring continuous operation since down time issignificantly reduced because filters do not generally need to beremoved and cleaned.

Tangential flow filtration can be used for several purposes includingconcentration and diafiltration, among others. Concentration is aprocess whereby solvent is removed from a solution while solutemolecules are retained. In order to effectively concentrate a sample, amembrane having a NMWL or MWCO that is substantially lower than themolecular weight of the solute molecules to be retained is used.Generally, one of skill may select a filter having a NMWL or MWCO ofthree to six times below the molecular weight of the target molecule(s).

Diafiltration is a fractionation process whereby small undesiredparticles are passed through a filter while larger desired molecules aremaintained in the retentate without changing the concentration of thosemolecules in solution. Diafiltration is often used to remove salts orreaction buffers from a solution. Diafiltration may be either continuousor discontinuous. In continuous diafiltration, a diafiltration solutionis added to the sample teed at the same rate that filtrate is generated.In discontinuous diafiltration, the solution is first diluted and thenconcentrated back to the starting concentration. Discontinuousdiafiltration may be repeated until a desired concentration of thesolute molecules is reached.

At least three process variables that are important in a typical TFFprocess: the transmembrane pressure, feed rate, and flow rate of thepermeate. The transmembrane pressure is the force that drives fluidthrough the fitter, carrying with it permeable molecules. In someembodiments, the transmembrane pressure is between 1 and 30 pounds persquare inch (psi), inclusive.

The feed rate (also known as the crossflow velocity) is the rate of thesolution flow through the feed channel and across the filter. The feedrate determines the force that sweeps away molecules that may otherwiseclog or foul the filter and thereby restrict filtrate flow. In someembodiments, the feed rate is between 50 and 500 mL/minute. In someembodiments, the feed rate is between 50 and 400 mL/minute. In someembodiments, the feed rate is between 50 and 300 mL/minute. In someembodiments, the feed rate is between 50 and 200 mL/minute. In someembodiments, the feed rate is between 75 and 200 mL/minute. In someembodiments, the feed rate is between 100 and 200 mL/minute. In someembodiments, the feed rate is between 125 and 175 mL/minute. In someembodiments, the feed rate is 130 mL/minute. In some embodiments, thefeed rate is between 60 mL/min and 220 mL/min. In some embodiments, thefeed rate is 60 mL/min or greater. In some embodiments, the feed rate is100 mL/min or greater. In some embodiments, the feed rate is 150 mL/minor greater. In some embodiments, the feed rate is 200 mL/min or greater.In some embodiments, the feed rate is 220 mL/min or greater.

The flow rate of the permeate is the rate at which the permeate isremoved from the system. For a constant feed rate, increasing permeateflow rates can increase the pressure across the filter, leading toenhanced filtration rates while also potentially increasing the risk offilter clogging or fouling. The principles, theory, and devices used forTFF are described in Michaels et al., “Tangential Flow Filtration” inSeparations Technology, Pharmaceutical and Biotechnology Applications(W. P. Olson, ed., Interpharm Press, Inc., Buffalo Grove, Ill. 1995).See also U.S. Pat. Nos. 5,256,294 and 5,490,937 for a description ofhigh-pertbrinance tangential flow filtration (HP-TFF), which representsan improvement to TFF. In some embodiments, the flow rate is between 10and 100 mL/minute. In some embodiments, the flow rate is between 10 and90 mL/minute. In some embodiments, the flow rate is between 10 and 80mL/minute. In some embodiments, the flow rate is between 10 and 70mL/minute. In some embodiments, the flow rate is between 10 and 60mL/minute. In some embodiments, the flow rate is between 10 and 50mL/minute. In some embodiments, the flow rate is between 10 and 40mL/minute. In some embodiments, the flow rate is between 20 and 40mL/minute. In some embodiments, the flow rate is 30 mL/minute.

Any combinations of various process variables described herein may beused. In some embodiments, the tangential flow filtration is performedat a feed rate of approximately 100-200 mL/minute (e.g., approximately100-180 mL/minute, 100-160 mL/minute, 100-140 mL/minute, 110-190mL/minute, 110-170 mL/minute, or 110-150 mL/minute) and/or a flow rateof approximately 10-50 mL/minute (e.g., approximately 10-40 mL/minute,10-30 mL/minute, 20-50 mL/minute, or 20-40 mL/minute). In someembodiments, the tangential flow filtration is performed at a feed rateof approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200 mL/minute and/or a flow rate of approximately 10, 20, 30, 40, or 50mL/minute.

Further flow rates to accommodate large (commercial) scale purificationwould entail the tangential flow filtration being performed at a feedrate of approximately 10 L-200 L/minute. (e.g., approximately 10-180L/minute, 100-160 L/minute, 100-140 L/minute, 110-190 L/minute, 110-170L/minute, or 110-150 L/minute) and/or a flow rate of approximately 10-50L/minute (e.g., approximately 10-40 L/minute, 10-30 L/minute, 20-50L/minute, or 20-40 L/minute). In some embodiments, the tangential flowfiltration is performed at a feed rate of approximately 100, 110, 120,130, 140, 150, 160, 170, 180, 190, or 200 L/minute and/or a flow rate ofapproximately 10, 20, 30, 40, or 50 L/minute.

As described above, filters used in TFF may have any of a variety ofpore sizes, and thus NMWLs. In some embodiments, a filter will have aNMWL of between 100 kDa and 1,000 kDa. In some embodiments, a filterwill have a NMWL of between 200 kDa and 700 kDa. In some embodiments, afilter will have a NMWL between 200 kDa and 500 kDa. In someembodiments, a filter has a NNTWL of 300 kDa. In some embodiments, afilter has a NMWL of 500 kDa.

In some embodiments, a tangential flow filtration according to theinvention is performed using only aqueous solvents. In some embodiments,a tangential flow filtration according to the invention is performedusing water as the solvent.

Characterization of Purified mRNA

In various embodiments, mRNA purified according to the present inventionis substantially free of impurities from mRNA synthesis processincluding, but not limited to, prematurely aborted RNA sequences, DNAtemplates, and/or enzyme reagents used in in vitro synthesis.

A particular advantage provided by the present invention is the abilityto remove or eliminate a high degree of prematurely aborted RNAsequences (also known as “shortmers”), In some embodiments, a methodaccording to the invention removes more than about 90%, 95%, 96%, 97%,98%, 99% or substantially all prematurely aborted RNA sequences. In someembodiments, mRNA purified according to the present invention issubstantially free of prematurely aborted RNA sequences. In someembodiments, mRNA purified according to the present invention containsless than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) ofprematurely aborted RNA sequences. In some embodiments, mRNA purifiedaccording to the present invention contains less than about 1% (e.g.,less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%)of prematurely aborted RNA sequences. In some embodiments, mRNA purifiedaccording to the present invention contains contains undetectableprematurely aborted RNA sequences as determined by, e.g., eithidiumbromide and/or Coomassic staining. In some embodiments, prematurelyaborted RNA sequences comprise less than 15 bases (e.g., less than 14,13, 12, 11, 10, 9 or 8 bases). In some embodiments, the prematurelyaborted RNA sequences comprise about 8-12 bases.

In some embodiments, a method according to the present invention removesor eliminates a high degree of enzyme reagents used in in vitrosynthesis including, but not limited to, T7 RNA polymerase, DNAse I,pyrophosphatase, and/or RNAse inhibitor. In some embodiments, thepresent invention is particularly effective to remove T7 RNA polymerase.In some embodiments, a method according to the invention removes morethan about 90%, 95%, 96%, 97%, 98%, 99% or substantially all enzymereagents used in in vitro synthesis including. In some embodiments, mRNApurified according to the present invention is substantially free ofenzyme reagents used in in vitro synthesis including. In someembodiments, MRNA purified according to the present invention containsless than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of enzymereagents used in in vitro synthesis including. In some embodiments, mRNApurified according to the present invention contains less than about 1%(e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%,or 0.1%) of enzyme reagents used in in vitro synthesis including. Insome embodiments, mRNA purified according to the present inventioncontains undetectable enzyme reagents used in in vitro synthesisincluding as determined by, e.g., ethidium bromide and/or Coomassiestaining.

In various embodiments, mRNA purified using a method described hereinmaintain high degree of integrity. As used herein, the term “mRNAintegrity” generally refers to the quality of mRNA after purification.In some embodiments, mRNA integrity refers to the percentage of mRNAthat is not degraded after tangential flow filtration. mRNA integritymay be determined using methods well known in the art, for example, byRNA agarose gel electrophoresis (e.g., Ausubel et al., John Weley &Sons, Inc., 1997, Current Protocols in Molecular Biology). In someembodiments, mRNA purified according to the present invention has anintegrity greater than about 95% (e.g., greater than about 96%, 97%,98%, 99% or more). In some embodiments, mRNA purified according to thepresent invention has an integrity greater than 98%. In someembodiments, mRNA purified according to the present invention has anintegrity greater than 99%. In some embodiments, mRNA purified accordingto the present invention has an integrity of approximately 100%.

EXAMPLES Example 1 Generation and Purification of Messenger RNA (mRNA

Synthesis of mRNA

In each of the examples below, the synthesis of mRNA was conducted undercomplete RNAse-free conditions. All tubes, vials, pipette tips,pipettes, buffers, etc. were required to be nuclease-free, unlessexplicitly stated otherwise.

101011 In the following examples, unless otherwise described, mRNA wassynthesized via in-vitro transcription from a linearized DNA template.To produce the desired mRNA pre-cursor (IVT) construct, a mixture of˜100 ug of linearized DNA, rNTPs (3.33 mM), DTT (10 mM), T7 RNApolymerase, RNAse inhibitor, Pyrophosphatase and reaction buffer (10×,800 mM Hopes (pH8.0), 20 mM Spermidine, 250 mM MgCl₂, pH 7.7) wasprepared with RNase-free water to a final volume of 2.24 mL. Thereaction mixture is incubated at 37° C. for a range of time between 20minutes-120 minutes. Upon completion, the mixture is treated with DNaseI for an additional 15 minutes and quenched accordingly.

Addition of 5′ Cap and 3′ Tail

The purified mRNA product from the aforementioned IVT step (and possiblyinitial TFF filtration as well) was denatured at 65° C. for 10 minutes.Separately, portions of GTP (20 mM), S-adenosyl methionine, RNAseinhibitor, 2′-O-Methyltransferase and guanylyl transferase are mixedtogether with reaction buffer (10×, 500 mM Tris-HCl (pH8.0), 60 mM KCl,12.5 mM MgCl₂) to a final concentration of 8.3 mL. Upon denaturation,the mRNA is cooled on ice and then added to the reaction mixture. Thecombined solution is incubated for a range of time at 37° C. for 20-90minutes. Upon completion, aliquots of ATP (20 mM), PolyA Polymerase andtailing reaction buffer (10×, 500 mM Tris-HCl (pH8.0), 2.5M NaCl, 100 mMMgCl₂) are added and the total reaction mixture is further incubated at37° C. for a range of time from 20-45 minutes. Upon completion, thefinal reaction mixture is quenched and purified accordingly.

Purification via Tangential Flow Filtration

In the following examples, unless otherwise described, the tangentialflow filtration (TFF) system consisted of a filtration membrane and aperistaltic pump (Millipore Labscale TFF system) with tangentialcirculation of the fluid across the membrane at a feed rate of ˜130mL/min with a 30 mL/min flow rate for the permeate. The TFF membraneemployed was a MidiKros 500 kDa mPES 115 cm² (Spectrum Labs). Beforeuse, the filter cartridge was washed with nuclease free water andfurther cleaned with 0.2N NaOH. Finally the system was cleaned withnuclease free water until the pH of permeate and retentate reached a pH

Example 2 Analysis of Purified mRNA

Testing for Presence of Enzymes in Purified mRNA

Unless otherwise described, standard Coomassie-stained protein gels wereperformed to determine the presence of any residual reagent enzymespresent before and after purifications. In some instances, BCA assayswere performed as well.

Assessment of mRNA Integrity via Agarose Gel Electrophoresis Assays

Unless otherwise described, messenger RNA size and integrity wereassessed gel electrophoresis. Either self-poured 1.0% agarose gel orInvitrogen E-Gel precast 1.2% agarose gels were employed. Messenger RNAwas loaded at 1.0-1.5 ug quantities per well. Upon completion, messengerRNA bands were visualized using ethidium bromide.

In Vitro mRNA Integrity Assays

Unless otherwise described, in vitro transfections of firefly luciferasemRNA were performed using HEK293T cells. Transfections of one microgramof each mRNA construct were performed in separate wells usinglipofectamine. Cells were harvested at select time points (e.g. 4 hour,8 hour, etc.) and respective protein production was analyzed. For FFLmRNA, cell lysates were analyzed for luciferase production viabioluminescence assays.

Bioluminescence Analysis

In examples including a fluorescent assessment of provided RNA, thebioluminescence assay was conducted using a Promega Luciferase AssaySystem (Item #E1500), unless otherwise specified. The Luciferase AssayReagent was prepared by adding 10 mL of Luciferase Assay Buffer toLuciferase Assay Substrate and mix via vortex. Approximately 20 uL ofhomogenate samples were loaded onto a 96-well plate followed by 20 uL ofplate control to each sample. Separately, 120 μL of Luciferase AssayReagent (prepared as described above) was added to each well of a96-well flat bottomed plate. Each plate was then inserted into theappropriate chambers using a Molecular Device Flex Station instrumentand measure the luminescence (measured in relative light units (RLU)).

Example 3 Generation and Purification of Firefly Luciferase (FFL)Messenger RNA (mRNA)

This example illustrates that, according to various embodiments, acombination of tangential flow filtration (TFF) and a denaturing agentmay be used according to provided methods to product a highly purifiedmRNA product. In this example, urea is used as the protein denaturingagent.

In this example, a five milligram batch of firefly luciferase (FFL) RNA(SEQ ID NO: 1, below) was transcribed via the in vitro methods describedabove to produce the aforementioned intermediate construct with no capand no polyA tail. This reaction maintained a total volume of 2.24 mLand was quenched upon completion by an equivalent volume of I OM urea,bringing the final urea concentration to 5M. The resultant solution wasincubated for five minutes at room temperature and transferred to thetangential flow filtration (TFF) system reservoir. The sample wasdiluted to 200 mL with nuclease free water and washed with 1200 mLnuclease free water by ultrafiltration of 200 mL at a time. Followingthis, the sample was treated with 200 mL 10 mM Sodium Citrate (pH 6.4)followed by 600 ml wash with nuclease free water. Finally the sample wasconcentrated to 2 mL and the final concentration was determined viaabsorption at 260 nm (λ_(max)).

Codon-Optimized Firefly Luciferase (FFL) mRNA (SEQ ID NO: 1) X₂AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCA GCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCC UGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGA GAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGC AGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAG CUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGU GAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUC AUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCALUUGCCACCCG GCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAG UAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAU GCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACC ACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUU CGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUA UUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCG GCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCA GGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCA GUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGA ACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUAC AAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCAC UUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGG AGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGG CGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAU GUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAG GACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAU CGCCGUGUAY ₂5′ and 3′ UTR Sequences: (SEQ ID NO: 2) X₂ = GGGAUCCUACC (SEQ ID NO: 3)Y₂ = UUUGAAUU

Approximately 5 mg of TFF-purified firefly luciferase RNA was capped andtailed in a final reaction volume of 9 mL, as described above. A portionof this reaction mixture (6.7 ml) was treated with 5M urea for 5 minutesat morn temperature (RT) and purified using TFF. Approximately 1.5 mg ofthe cap/tail reaction mixture was purified via TFF using solely waterand isolated. Separately, another small portion of the cap/tail reactionmixture was purified using a Qiagen RNeasy Purification kit according topublished protocol. The three isolated final mRNA batches werealiquotted and transfected into HEK293T cells as described below. Celllysates were analyzed for the presence of FFL protein via fluorescencedetection (FFL activity).

In this example, in order to remove reaction enzymes in this example, aportion of the FFL mRNA IVT reaction mixture was subjected to 10M urearesulting in a final concentration of 5M urea. This solution wasincubated for five minutes at room temperature and then purified via TFFas described above. FIG. 1 shows a coomassie stained protein gel whichshows the resulting mRNA isolated after TFF employing the aforementionedurea conditions. There is no detectable enzyme present upon completion.

After producing the capped and tailed FFL mRNA product, TFF methods wereemployed further to purify the final target mRNA. Portions of the samecap/tail reaction mixture were separately aliquotted and purified eithervia TFF with no urea or via spin-column methods (Qiagen RNeasy Kit) forcomparison. A comparison of the final mRNA isolated either by TFF orspin column was made using gel electrophoresis and is depicted in FIG. 2. Further, residual enzyme levels were monitored via protein gel (FIG. 3). In FIG. 2 , one cart clearly see the respective “IVT” FFL mRNA bandsmigrating at ˜1900 nt with the capped & tailed (C/T) final mRNAapproximately 2100 nt long. The “shortmer” band typically observed usingspin-column isolation after the cap/tail step is indeed observed in Lane4.

It is apparent that the shortmer band is not present after the cap/tailstep when TFF-purified mRNA is employed. While substantial amounts ofenzyme reagents can be removed using either purification method,shortmer impurities cannot. This demonstrated that the tangential flowfiltration methods described herein are a successful and efficientmethod for purification of prematurely aborted sequences during mRNAtranscription.

In order to determine whether provided mRNA can be translated into thedesired protein, a comparison of each of the isolated FFL mRNAconstructs (-ITT vs spin-column) was made. Each of the three constructslisted below were transfected into HEK293T cells and corresponding FFLprotein production was assessed via FFL protein activity in the form ofFFL luminescence upon exposure to luciferin (vada supra).

FFL Constructs:

1. FFL IVT purified via TFF (urea) and C/T step via TFF (no urea)

2. FFL IVT purified via TFF (urea) and C/T step via TFF (urea)

3. FFL IVT purified via spin column and C/T step via spin column

A comparison of luminescence output of FFL protein produced from each isrepresented in FIG. 4 . The integrity of the TFF-purified FFL mRNA ismaintained throughout the tangential flow filtration process under theconditions described (exposure to 5M urea).

Example 4 Generation and Purification of Factor IX (FIX) mRNA

This example further illustrates that, according to various embodiments,a combination of tangential flow filtration (TFF) and a denaturing agentmay be used according to provided methods to product a highly purifiedmRNA product. In this example, guanidinium thiocyanate is used as theprotein denaturing agent.

In this example, a second species of mRNA was produced and purified,this time coding for Factor IX (SEQ ID NO: 4, below). Initially, a fivemilligram batch of Factor IX (FIX) RNA was transcribed via in vitromethods as described above to produce the aforementioned RNA with no capand no polyA tail. This reaction maintained a total volume of 2.24 mLand was quenched upon completion by the addition of Proteinase K (4mg/ml IVT reaction) which was incubated in the reaction mixture at 37°C. for 5 minutes. Upon completion, 6M guanidinium thiocyanate (4.3 mL,final 4M) was added and the resultant solution was incubated for fiveminutes at room temperature and transferred to the TFF system reservoir.The sample was diluted to 200 mL nuclease free water and washed with1600 mL nuclease free water by ultrafiltration of 200 mL at a time. Uponcompletion, the sample was concentrated to ˜2 mL and the finalconcentration was determined via absorption at 260 nm (λ_(max)).

Human Factor IX (FIX) mRNA (SEQ ID NO: 4) X₁AUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCA CCAGGCCUCAUCACCAUCUGCCUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUG AAAACGCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAGUUUGUUCAAGG GAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAAGAAGCACGAGAAGLUUUUGAAAACACU GAAAGAACAACUGAAUUUUGGAAGCAGUAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUG GCGGCAGUUGCAAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAGAACUG UGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUUUGUAAAAAUAGUGCUGAUAAC AAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGACUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGC CAUUUCCAUGUGGAAGAGUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGA UGUGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAAGCACCCAAUCAUUU AAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAACCAGGUCAAUUCCCUUGGCAGGUUGUUUUGA AUGGUAAAGUUGAUGCAUUCUGUGGAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUG UGUUGAAACUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAACAUACAGAG CAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUGCAGCUAUUAAUAAGUACAACCAUG ACAUUGCCCUUCUGGAACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGA CAAGGAAUACACGAACAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCAC AAAGGGAGAUCAGCUUUAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCGAGCCACAUGUCUUCGAU CUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCUGGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCA AGGAGAUAGUGGGGGACCCCAUGUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGG GGUGAAGAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUCAACUGGAUUA AGGAAAAAACAAAGCUCACUUAAY ₁5′ and 3′ UTR Sequences: (SEQ ID NO: 5) X₁ =GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUU UGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUU CCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQ ID NO: 6)  Y₁ =  CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCAC CAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC

The above method was also performed as described above, with theaddition of actinomycin D (10 μg/ml IVT reaction) during the ProteinaseK step. By quenching the IVT reaction with Proteinase K (with or withoutactinomycin D), one can also successfully achieve removal of all enzymes(FIG. 5 ). While Proteinase K may facilitate removal, large scalemanufacturing of an mRNA drug substance would require this enzyme to bemade at large scale incurring additional unnecessary costs, andtherefore may not be a desired approach in some embodiments. As shown inFIG. 6 , FIX mRNA produced as described above (with and withoutactinomycin D), as well as FIX mRNA purified using 5M urea, does notcontain detectable levels of shortmers, similar to the results for FFLmRNA as described in Example 3.

Example 5 Generation and Purification of Cystic Fibrosis TransmembraneConductance Regulator (CFTR) mRNA

This example further illustrates that, according to various embodiments,a combination of tangential flow filtration (TFF) and a denaturing agentmay be used according to provided methods to product a highly purifiedmRNA product. In this example, potassium chloride is used as the proteindenaturing agent.

In this example, a third species of mRNA was produced and purified, thistime coding for the Cystic Fibrosis Transmembrane Conductance Regulator(CFTR, SEQ ID NO: 7, below). Initially, a five milligram batch of CFTRRNA was transcribed via in vitro methods as described above to producethe aforementioned RNA with no cap and no polyA tail. This reactionmaintains a total volume of 2.24 mL and was quenched upon completion byaddition of 2M KCl (200 mL). The resultant solution was incubated forfive minutes at room temperature and transferred to the TFF systemreservoir. The sample was diafiltrated at a constant volume of 200 mLwith 2M KCL in nuclease-free water for three to four diavolumes. Afterthis time, the resultant solution was washed with 400 mL nuclease-freewater by ultrafiltration of 200 mL at a time. Following this, the samplewas treated with 200 mL 1 mM Sodium Citrate (pH6.4) followed by 600 mlwash with nuclease free water. Finally, the sample was concentrated to 2mL and the final concentration was determined via absorption at 260 nm(λ_(max)).

Codon-Optimized Cystic Fibrosis TransmembraneConductance Regulator (CFTR) mRNA (SEQ ID NO: 7) X₁AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUC GUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGU CUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGA CCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUC AUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCA UUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUG UUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAG AUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGA UUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGC ACAUUUCGUGUGGAUUGCCCCGUUGGAAGUCGCCCUUUUGAUGGGGCUUAUUUGGGAGCUGUUGCAGGCA UCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGA UGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAA CAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACU GAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGU UCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCAC CACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGG UAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGU ACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGULUUGGAGAACU GUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCG AACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCG CGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAG CGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACC AUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCC AGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUU GUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUU GAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUA UGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAU CCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCA AGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAA CGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAA GCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUC UCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCA GGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGG ACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAA AACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAAC UCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGA UUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGU UACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCG CAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUC AAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGA GUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCU CCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAA GGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUaUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAU CUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUAC AUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCAC AGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUU1ACGCAUCUUGUGACCAGUUUGAAGGG AUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUC CAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUG UGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAI RLAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGA UAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAA AGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGG AUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAA CGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGG UCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACG GCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUU UAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAA GUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAG AUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGC GAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACA CUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCC AGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAG AUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAG UGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUU AAY ₁ 5′ and 3′ UTR Sequences:(SEQ ID NO: 5) X₁ = GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCC UCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 6) Y₁ =CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUC UCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC

In this example, in order to remove reaction enzymes, 2M KCldiafiltration was used. Exposure to large volumes of 2M KCl resulted insuccessful removal of all enzymes present in the reaction mixture(including T7 polymerase) as determined via protein gel electrophoresis(FIG. 7 ). As shown is agarose gel electrophoresis, the target messengerRNA remains intact after exposure to such conditions (FIG. 8 ).

Further, upon capping and tailing of the CFTR IVT construct, one cansuccessfully purify the final CFTR transcript (capped and tailed) viaTFF using 2M KCl. When comparing this final isolated product to the sameproduct purified via spin-column methods, one observes a greatlydiminished “shortmer” band as determined via gel electrophoresis (FIG. 9).

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to he limited to the above Description, butrather is as set forth in the following claims:

1-31. (canceled)
 32. A method of manufacturing messenger RNA (mRNA)comprising: (a) synthesizing mRNA in vitro to provide an impurepreparation of mRNA; and (b) purifying the mRNA; wherein purifying themRNA comprises: (i) subjecting the impure preparation of mRNA to adenaturing condition; and (ii) subjecting the treated impure preparationfrom step (b)(i) to tangential flow filtration.
 33. The method of claim32, wherein the in vitro synthesized mRNA is greater than about 1 kb,1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length. 34.The method of claim 32, wherein the in vitro synthesized mRNA comprisesone or more modifications to enhance stability.
 35. The method of claim34, wherein the one or more modifications are selected from modifiednucleotide, modified sugar phosphate backbones, 5′ and/or 3′untranslated region.
 36. The method of claim 32, wherein the in vitrosynthesized mRNA is unmodified.
 37. The method of claim 32, wherein thedenaturing condition is achieved through adding one or more denaturingagents selected from an enzyme, an acid, a solvent, a cross-linkingagent, a chaotropic agent, and/or high salt.
 38. The method of claim 37,wherein the impure preparation is incubated with the one or moredenaturing agents at a temperature below room temperature.
 39. Themethod of claim 37, wherein the impure preparation is incubated with theone or more denaturing agents at a temperature above room temperature.40. The method of claim 32, wherein the tangential flow filtration isperformed before a cap and poly-A tail are added to the in vitrosynthesized mRNA.
 41. The method of claim 32, wherein the tangentialflow filtration is performed after a cap and poly-A tail are added tothe in vitro synthesized mRNA.
 42. The method of claim 32, wherein thetangential flow filtration is performed both before and after a cap anda poly-A tail are added to the in vitro synthesized mRNA.
 43. The methodof claim 32, wherein the feed rate is between 50 mL/minute and 500mL/minute.
 44. The method of claim 32, wherein the feed rate is between10 L/minute and 200 L/minute.
 45. The method of claim 32, wherein theflow rate of the permeate is between 10 mL/minute and 100 mL/minute.